Conventional methods for transforming rice include electroporation method and PEG method using protoplasts, and these methods have been applied to Japonica rice which may easily be cultured. However, these methods may be applied only to the varieties for which redifferentiation system from protoplasts have been established, and have scarcely been applied to Indica rice which is difficult to culture.
Since the particle gun method does not need a protoplast-culturing system and so it can be applied to various varieties, the method has been more and more used in a number of laboratories. In general, it is thought that Indica rice varieties, especially those belonging to the so called Group I (Glaszmann J. C. (1987) Isozymes and classification of Asian rice varieties. Theor. Appl. Genet. 74:21-30) which occupies most part of the Indica rice varieties, are difficult to culture. However, the transformation efficiency of the varieties belonging to Group I by the particle gun method reported by Christou et.al. (Christou P., Ford, T. L. and Kofron, M. (1992) The development of a variety-independent gene-transfer method for rice. TIB TECH 10: 239-246), is as low as 2 to 3% per immature embryo. According to the recent report by other groups too, a transformation system with a high efficiency has not been obtained (LiL., Rongda, Q., Kochko, A., Fauquet, C. and Beachy, R. N. (1993), An improved rice transformation system using the biolistic method. Plant Cell Report 12: 250-255).
On the other hand, the Agrobacterium method has been widely used for dicotyledons as a simple and stable transformation method. However, it was thought that the Agrobacterium method could not be applied to monocotyledons such as rice (Potrykus I., (1990) Gene transfer to cereals: an assessment Bio/technology 8:535-542). Recently, it has been proved that the Agrobacterium method may be applied to rice which is a monocotyledon (WO94/00977; WO95/06722; Hiei Y., Ohta, S., Komari, T. and Kumashiro, T. (1994) Efficient transformation of rice (Oryza Sativa L.) mediated by transformation by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal 6:271-282), so that future development of this method as a useful transformation method is expected.
On the other hand, Rance et al. disclose an NB medium useful for inducing a callus having redifferentiation ability from mature seeds of Indica rice (Iann M. Rance,I. M. et al., Partial desiccation of mature embryo-derived calli, a simple treatment that dramatically enhances the regeneration ability of Indica rice, Plant Cell Reports (1994) 13:647-651). However, they did not investigate the effect of the NB medium on the selection of the transformed cells. Li et al. reported transformation of Japonica rice with high efficiency using a medium similar to NB medium (not containing NAA, BA and L-glutamine) (Li L. et al., (1993) An improved rice transformation system using the biolistic method. Plant Cell Report 12: 250-255). However, they reported that transformants of Indica rice were not obtained with a high efficiency. Further, Li et al. did not study application of the medium to the Agrobacterium method.
As mentioned above, the methods in which transformants are prepared from protoplasts have a problem that they cannot be applied to the varieties for which a regeneration system from protoplasts has not been established. As for the particle gun method, the transformation efficiencies of the reported methods for the varieties which are difficult to culture, such as the varieties belonging to Indica rice, are low.
Thus, it is thought that the Agrobacterium method may be a candidate for the method for transforming Indica rice. As mentioned above, transformation of Japonica rice by the Agrobacterium method is known. The present inventors investigated whether the method applied to Japonica rice may be applied to Indica rice or not.
The first candidate for the method for transforming rice by Agrobacterium is the method using a dedifferentiated tissue as described in WO94/00977 and Hiei et al. (1994). Thus, the present inventors tried to introduce gene by Agrobacterium into a callus, using several varieties of Indica rice belonging to Group I. As a result, it was proved that transformants could be obtained although the number was small. However, a transformation system having reproducibility could not be established. In cases where transformation is performed on a callus, it is necessary to employ a callus having a high cell-dividing ability and high regeneration ability. However, for rice varieties which are difficult to culture, it is not easy to induce a callus having high cell-dividing activity, which is suited for introduction of a gene. Therefore, it is thought that in cases where a callus is used as the sample tissue, the varieties to which this method can be applied is limited, and transformants cannot be obtained easily for the varieties which are difficult to culture.
As a method employing a tissue other than callus, it is thought that the method employing an immature embryo may be applied. However, if the method described in WO95/06722 or EP-A-0 672 752, which is effective for Japonica rice, is applied to Indica rice as it is, the transformation efficiency was low, so that a practical transformation system could not be established.